This invention relates to use of peptides which target the human transferrin receptor. Peptides of the invention can be used to direct other peptides, proteins and other diagnostic or therapeutic agents into cells for both diagnostic and therapeutic purposes.
Previous work relating to redirecting viral vectors in gene therapy by using short peptide ligands to redirect virus particles to specific cell types are known. One of the limitations of this strategy is that short peptide sequences that bind efficiently to cell surface receptors on specific cell types must be identified. One experimental approach to identify such short peptides that holds promise is bacteriophage display.
For more than a decade, phage display has exploited the physical linkage between random peptide sequences expressing on phage and the DNA encoding that sequence. This linkage allows for rapid identification of peptide ligands. A random peptide sequence is expressed as a fusion with a bacteriophage coat protein and is available for testing as a ligand for various targets. Phage display has successfully been used to identify single chain antibodies with specificity for various biological molecules. Phage display strategies can be used to elucidate the amino acids responsible for proteinxe2x80x94protein interactions, to find organ-specific phage, and to find substrate recognition sequences for enzymes. The process of using multiple rounds of phage display to enrich for a particular sequence is called biopanning.
The human transferrin receptor (hTfR) has been studied extensively as a model system for receptor-mediated endocytosis, a marker for cellular proliferation, and a target for therapeutics. The hTfR is ubiquitously expressed and over-expressed at least 100 fold in oral, liver, pancreatic, prostate and other cancers. This increase in transferrin receptor (TfR) in cancers has been attributed to the increased metabolism of these transformed cells, making the hTfR a useful diagnostic marker. Because of its expression pattern and pathway characteristics, the hTfR is an attractive target for therapeutics. The TfR is a dimer composed of two identical 95 kDa subunits and is responsible for the majority of cellular iron uptake. The type II cell surface receptor binds 80 kDa transferrin (Tf) and the complex is internalized through clathrin-coated pits. Iron is released from transferrin in the acidic early endosome and the apotransferrin-receptor complex is recycled back to the cell surface where apotransferrin is recycled.
A blast search failed to yield any significant homologies between either HAIYPRH (Seq. ID No. 1) or THRPPMWSPVWP (Seq. ID No. 2) to known proteins, including Tf.
This invention relates to peptides which are capable of binding to and internalizing with the human transferrin receptor (hTfR). The sequences HAIYPRH (Seq. ID No. 1) and THRPPMWSPVWP (Seq. ID No. 2) are capable of binding to and internalizing with the human transferrin receptor. When these molecules were fused with other molecules, the fusion product was internalized in cells expressing hTfR. The sequences have use for targeting other peptides and proteins into cells expressing hTfR. The phage display system using whole cell selective biopanning could also be applied to find small ligands for other cell surface receptors. This sequence is not found in human transferrin protein. Furthermore, this sequence does not compete with transferrin itself for binding to the hTfR.
It is important that easily produced peptides that can facilitate entry of diagnostically and therapeutically useful peptides and proteins into cells having particular characteristics be available. The identification of peptides that will facilitate entry of such peptides into cells which are more likely to be aberrant has particular use. The peptides of the invention are useful for facilitating entry of diagnostically and therapeutically useful agents, including peptides and proteins. Since malignant cells produce increased expression of hTfR, the peptides, HAIYPRH (Seq. ID No. 1) and THRPPMWSPVWP (Seq. ID No. 2), are particularly useful for study and treatment of malignancies.
A phage display selection strategy was utilized that resulted in identification of the peptides. This selection system is based on alternating rounds of negative selection on chicken embryo fibroblast (CEF) cells lacking hTfR and positive selection on chicken embryo fibroblast cells expressing hTfR (CEF+hTfR). Biopanning on whole cells was exploited to select the peptides HAIYPRH (Seq. ID No. 1) and THRPPMWSPVWP (Seq. ID No. 2). These peptides were able to target a macromolecule to and internalize through the hTfR, as was demonstrated by phage binding, competition and immunofluorescence studies. It was also shown that these two peptides bind sites that do not overlap with the native ligand, transferrin, indicating they could be used in vivo for targeting macromolecules to the endocytic pathway in hTfR-positive cells.
The biopanning procedure could be applied to find small peptide ligands for other cell surface receptors. There is a great need to find new epitopes on various cancer cell types for diagnostic purposes. The subtractive method of biopanning disclosed herein would be useful for finding new cell surface markers. Biopanning on whole cells can be especially useful in situations where the receptor can not be purified or does not maintain its native confirmation when isolated.
Materials and Methods:
Cell lines: The two chicken embryo fibroblast cell lines, CEF and CEF+hTfR, used for selective biopanning, were described previously (Collawn, et al, Cell, 63, 1061-1072 (1990) and Odorizzi, et al., J. Cell Biol., 126, 317-330 (1994)). Chicken embryo fibroblasts have been used extensively for study of hTfR. The native cells express chicken transferrin receptors, but this receptor cannot bind human transferrin. Two cell lines were previously established through stable transduction with retroviral vectors to yield CEF and CEF+hTfR cells. CEF cells do not express the human transferrin receptor. CEF+hTfR cells constitutively express hTfR. Protein expression of hTfR by CEF cells was periodically checked by 125I-Tf binding. Both cells are grown in monolayer cultures in Dulbecco""s Modified Eagle Medium supplemented with 1% chicken serum, 1% bovine calf serum, 1% L-glutamine 200 nM, and 2% tryptose phosphate and maintained at 37xc2x0 C. in 13% CO2.
Antibodies: Monoclonal anti-GFP (green fluorescent protein) antibody (Clontech, Palo Alto, Calif.) was used for Western blot analysis and immunofluorescence at 1:5,000 and 1:250 dilution, respectively. Horse radish peroxidase conjugated goat anti-mouse antibody (Pierce, Rockford, Ill.), Oregon-Green and Texas-Red secondary antibodies (Molecular Probes, Eugene, Oreg.) were used at 1:10,000, 1::250, 1:250 dilution, respectively.
Electrophoretic Methods: Samples were dissolved on SDS-PAGE gels by the methods of Laemmli and transferred to nitrocellulose membrane by electroblotting for Western blot analysis (Laemmli, U.K, Nature, 227, 680-685). The membranes were blocked with 5% milk in tris buffered saline with 1% Trition X-100 (TBS-TX) (50 mM Tris-HCL, pH 7.5, 0.2 M NaCl, 1% Triton X-100), and incubated with primary antibody in TBS-TX with 2.5% milk overnight at 4xc2x0 C. The membranes were then washed in TBS-TX and incubated with peroxidase-conjugated secondary antibody and developed with the enhanced chemiluminescence (ECL) kit in accord with the manufacturer""s instructions (Amersham Pharmacia Biotech, Buckinghamshire, England).
Biopanning: Ph.D.-7(trademark) or Ph.D.-12(trademark) Phage Display Peptide Library Kit (New England Biolabs, Inc, Bevery, Mass.) was used for biopanning on CEF and CEF+hTfR cells. The Ph.D.(trademark) phage display peptide library is based on a combinatorial library of random 7 or 12 amino acid peptides fused to a minor coat protein of the filamentous coliphage M13. In separate studies, two different phage display peptide libraries were used to select for 7-mer and 12 mer peptide sequences that could bind the hTfR expressed on the surface of CEF+hTfR cells. Cells were washed and incubated in serum-free opti-MEM (Gibco BRL Life Technologies, Gaithersburg, Md.) at 37xc2x0 C. for 1 hour prior to all biopanning procedures. Phage binding was carried out at 4xc2x0 C. in serum-free Opti-MEM with 1xc3x97106 cell/3.5 cm well. Initial biopanning procedures applied 2xc3x971011 phage to CEF cells for two hours; unbound phage were transferred to CEF+hTfR cells for 1 hour. Cells were washed 10 times with Opti-MEM, and bound phage was quickly eluted with low pH buffer (0.2 M glycine-HCL, pH 2.2) and neutralized with 1M Tris-HCl, pH 9.1. Eluted phage were amplified in 20 ml Luria-Bertani medium (LB) containing E. coli ER2537 (for 7-mer phage) and ER2783 (for 12-mer phage) at 37xc2x0 C. Phage from liquid cultures were obtained by clearing the supernatant twice by centrifugation at 10,000 rpm for 15 minutes at 4xc2x0 C., and precipitated with 1/6 volume of PEG/NaCl (10% polyethylene glycol-8000, 2.5M NaCl) at 4xc2x0 C. overnight. Phage pellets were suspended in 1 ml TBS (50 mM Tris-HCl, 150 mM NaCl), and precipitated with PEG/NaCl for 1 hour. Amplified phage were resuspended with 200 xcexcl TBS, 0.02% NaN3, and these amplified phage were used for additional rounds of biopanning. After each round of biopanning, the final elutes were titrated, amplified in E. coli, and plated onto LB plates. The plates were incubated at 37xc2x0 C. overnight. Individual plaques were subjected to plaque amplification, DNA purification, and DNA sequencing using a modified Sanger sequencing reaction (Sanger, et al., Pro Natl Acad Sci USA, 74, 5463-5467 (1977)) with the appropriate sequencing primers.
Peptide Synthesis: The peptides HAIYPRH (Seq. ID No. 1), IRHPHYA (Seq. ID No. 3), THRPPMWSPVWP (Seq. ID No. 2), and PWRPSHPVWMPT (Seq. ID No. 4) were synthesized on an Applied Biosystems Model 440 by means of the solid phase peptide synthesis procedure at the Peptide Synthesis Core Facility of the University of Alabama at Birmingham (UAB) Comprehensive Cancer Center. These peptides were purified by high pressure liquid chromatography, and the molecular weights were confirmed by mass spectrometry.
Binding and Competition Studies: Purified phage populations were amplified and were verified to be homogenous through DNA sequencing. Cells were prepared for binding as was described for biopanning procedures. Preparations of plaque-purified and titered phage (1xc3x971011) were incubated in serum-free Opti-MEM on either CEF or CEF+hTfR cells at 4xc2x0 C. for 1 hour. The cells were washed repeatedly with Opti-HEM and bound phage were eluted with low pH buffer and subsequently titered. In competition studies, holo-transferrin (Calbiochem, La Jolla, Calif.) or synthesized peptides were added to CEF+hTfR cells prior to addition of the phage for 1 hour at 4xc2x0 C. Multiple trials were completed and average titers and standard deviations determined. The titers determined on CEF+hTfR cells were divided by the titers determined on CEF cells and multiplied by 100 to yield fold over control data points.
Modified GFP Constructs: Transferrin from human serum, bovine serum albumin (BSA), and purified wild-type GFP (wtGFP) were obtained from Sigma (St. Louis, Mo.) and Clontech (Palo Alto, Calif.), respectively. The tagged GFP genes were generated by the PCR with template DNA Clontech""s GFP vector. The PCR reactions were carried out in a Perkin Elmer Cetus DNA Thermal Cycler for 30 cycles of 95xc2x0 C., 1 minute; 55xc2x0 C., 1 minute; and 72xc2x0 C., 1 minute.
The PCR products were purified with the Qiagen Gel Extraction Kit (Qiagen, Valencia, Calif.) and cut with BcrlII and HindIII restriction enzymes (Roche, Nutley, N.J.), and subcloned into the pET-32a(+) bacterial expression vector (Novagen, Madison, Wis.). The resulting expression vector was verified using a modified Sanger sequencing method. The tagged GFP expression plasmids were transformed into BL21/DE3 E. coli and expression was induced for 3-4 hours with 1 mM isopropyl-xcex2-D-thioglactopyranoside (IPTG) when the culture O.D.,600=0.5. Cells were pelleted, then resuspended in phosphate buffer with 20 mM imidazole followed by passage through a French press at 10,000-15,000 psi. Cell lysates were passed over a PisTrap nickel column (Amersham Pharmacia Biotech, Piscataway, N.Y.). The column was washed and finally eluted using an imidazole gradient. The purified protein was assayed by SDS-PAGE followed by Coomassie staining and Western blot analysis with a GFP monoclonal antibody (Clontech). ECL development was carried out as per the manufacturer""s instructions (Amersham Pharmacia Biotech). Protein concentrations were determined with the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, Calif.).
Immunofluorescence: CEF+hTfR cells were grown on glass coverslips to 50% to 75% confluence. The coverslips were washed and incubated in serum-free Opti-MEM media at 37xc2x0 C. for 1 hour. Then 2 xcexcg of wild-type GFP (Clontech), HAIYPRH (Seq. ID No. 1)-tagged GFP, THRPPMWSPVWP (Seq. ID No. 2)-tagged GFP, or Texas-Red Tf (Molecular Probes) was applied to cells in serum-free Opti-MEM media for 1 hour at 4xc2x0 C. or 37xc2x0 C. Cells were washed with Opti-MEM, then fixed in 3% formaldehyde for 30 minutes at 4xc2x0 C. Alternatively, the cells were acid-washed with 0.2 M glycine-HCl, Ph 2.2, prior to fixation. A GFP monoclonal antibody (Clontech) was used in conjunction with an Oregon-Green Goat Anti-Mouse (Molecular Probes) to augment GFP fluorescence. All slides were counterstained with DAPI (2(4Amidinophenyl)-6indole carbamidinedihydrochioride) (Sigma). The microscopic slides were mounted in Prolong(trademark) antifade medium (Molecular Probes). Images were captured on an AX70 microscope with Olympus Camera (Olympus, Melville, N.Y.) and analyzed with ESPRIT software (Life Science Resources, Cambridge, England). Final figures were assembled using Microsoft Power Point (Microsoft Corp., Redmond, Wash.). For colocalization studies, CEF+hTfR were incubated with 2 xcexcg/ml GFP fusion protein and 2 xcexcg/ml of Texas-Red Tf for 1 hour and processed as described above.
Standard Analysis: Purified proteins (transferrin, wtGFP, GFP-HAIYPRH (Seq. ID No. 1) and GFP-THRPPMWSPVWP (Seq. ID No. 2) were labelled with 125I to a specific activity of 1-2 xcexcCi/xcexcg with CPM/xcexcg determined by a gamma counter and Bradford assay. CEF+hTfR cells were plated in duplicate at a density of 7.5xc3x97104 cells/well in 24 well dishes and grown overnight. Cells were washed and incubated in serum-free Opti-MEM for 1 hour at 37xc2x0 C. Cells were placed on ice with the various amounts of labelled protein in a total of 200 xcexcl of cold 0.1% BSP in phosphate buffered saline (PBS). After 1 hour, the unbound protein was removed and cells were washed 4 times with 0.1% BSA in PBS. 1M NaOH was added to lyse the cells for determining the bound fraction. Both unbound and bound fractions were counted in a gamma counter and binding affinities were determined using Scatchard analysis. Studies were repeated 3 times and yielded comparable binding affinities for all proteins tested.